The present invention is based on the discovery of a new N-terminal region processed form of human platelet factor-4, hereinafter modified platelet factor-4 (MPF-4). The invention also includes a proteolytically cleaved, non-reduced form of platelet factor-4, hereinafter cleaved platelet factor-4 (CPF-4). Both MPF-4 and CPF-4 may be isolated from the spent culture medium of lipopolysaccharide-stimulated peripheral blood leukocytes (PBLs).
Amino acid sequencing revealed that MPF-4 is homologous to platelet factor-4 (PF-4) beginning with Ser-17 and thus is presumed to be a naturally-occurring cleavage product of PF-4. CPF-4 bears the same amino acid sequence as PF-4 but differs in that the peptide bond between residues 16 and 17 is absent. However, the two resulting peptides remain bonded via disulfide bridges. Most significantly, the compounds of the present invention are potent inhibitors of endothelial cell proliferation and are anywhere from 10-100 fold more active than native PF-4, depending on the source of PF-4 and the reporting laboratory.
PF-4 is the prototype member of a growing family of small inducible proteins that are released from various cell types after stimulation with mitogens or cytokines. This family of proteins, known as "intercrines," has been found to modulate a variety of biological processes such as angiogenesis, cell proliferation, coagulation, inflammation, and tissue repair. See Oppenheim et al., Properties of the Novel Proinflammatory Supergene "Intercrine" Cytokine Family, Ann. Rev. Immunol. 9: 617-648, 1991; Taylor et al., Protamine is an Inhibitor of Angiogenesis, Nature 297: 307-312, 1982; and Maione et al., Inhibition of Angiogenesis by Recombinant Human Platelet Factor-4 and Related Peptides, Science 247: 77-79, 1990).
Other members of the intercrine family include interluekin-8 (IL-8), melanocyte growth stimulating activity (hGro/MGSA), .beta.-thromboglobulin (.beta.-TG), neutrophil activating protein (NAP-2), IP-10, and macrophage inflammatory protein (MIP-2). See Lindley et al., Synthesis and Expression in Escherichia coli of the Gene Encoding Monocyte-derived Neutrophil-Activating Factor: Biological Equivalence between Natural and Recombinant Neutrophil-activating Factor, Proc. Natl. Acad. Sci. 85: 9199-9203, 1988; Walz et al., A Novel Cleavage Product of B-Thromboglobulin Formed in Cultures of Stimulated Mononuclear Cells Activates Human Neutrophils, Biochem. Biophys. Res. Commun. 159: 969-975, 1989; Luster et al., Gamma-interferon Transcriptionally Regulates an Early Response Gene Containing Homology to Platelet Proteins, Nature 315: 672-676, 1985; and Wolpe et al., Identification and Characterization of Macrophage Inflammatory Protein 2, Proc. Natl. Acad. Sci. 86: 612-616, 1989).
The complete primary structure of PF-4 is well known in the art (Poncz et al., Cloning and Characterization of Platelet Factor 4 cDNA Derived from a Human Erythroleukemic Cell Line, Blood 69: 219-223, 1987. Analogs and fragments of PF-4 are also well known and have been referred to as "Oncostatin-A in U.S. Pat. Nos. 4,645,828 and 4,737,580, herein incorporated by reference. Studies have shown that the intercrine family of proteins contain a characteristic cysteine-X-cysteine (CXC) motif located in the N-terminal region. The CXC motif participates in producing the secondary and tertiary structure of native PF-4 via formation of intramolecular disulfide bonds with residues Cys-36 and Cys-51 (St. Charles et al., The Three-dimensional Structure of Bovine Platelet Factor 4 at 3.0-A Resolution, J. Biol. Chem. 264: 2092-2098, 1989).
Moreover, based on the published three dimensional structure of bovine PF-4, it was determined that N-terminal residues Gln-9 to Val-19 form a large open loop and that Thr-16 hydrogen bonds to Cys-51. These N-terminal structures have been shown to be important for the immunoregulatory activity of PF-4 (Katz et al., Protease-induced Immunoregulatory Activity of Platelet Factor 4, Proc. Natl. Acad. Sci. 83: 3491-3495, 1986).
Although PF-4 is mainly found within the alpha-granules of platelets, genomic cloning has revealed evidence for duplication of the human PF-4 gene producing alternative forms of PF-4, namely PF-4varI and PF-4alt (Doi, et al., Structure of the Rat Platelet Factor 4 Gene: A Marker for Magakaryocyte Differentiation, Mol. Cell Biol. 7: 898-912. 1987). The deduced amino acid sequence of the variants shows important differences in the N-terminal leader sequence and in the lysine-rich C-terminal domain (Green et al., Identification and Characterization of PF4var1, a Human Gene Variant of Platelet Factor 4, Mol. Cell. Biol. 9: 1445-1451, 1989; Eisman et al., Structural and Functional Comparison of the Genes for Human Platelet Factor 4 and PF4alt, Blood 76: 336-344, 1990). The changes in the leader sequence suggests a difference in its mode of secretion and the tissue type where it is expressed. Thus, the alternate forms of PF-4 may also be produced by cells other then platelets.
It is also well known that the lysine rich C-terminal region of PF-4 strongly binds to heparin and related glycosaminoglycans (Rucinski et al., Human Platelet Factor 4 and its C-terminal Peptides: Heparin Binding and Clearance from the Circulation, Thrombos. Haemostas. 63: 493-498, 1990).
Mutant forms of PF-4 have been disclosed as have methods of using the mutants for treating angiogenic diseases. See U.S. Pat. Nos. 5,086,164 and 5,112,946, herein incorporated by reference. These patents teach PF-4 modifications made in the C-terminal region resulting in analogs that no longer have heparin-binding capacity. The present invention discloses MPF-4 and CPF-4 which are processed forms of PF-4.
Finally, currently held beliefs in medical science state that angiogenesis is required for solid tumors to grow beyond several cubic centimeters. Therefore, the use of angiogenic inhibitors such as MPF-4 and CPF-4 presents a potentially viable approach for the treatment of solid tumors.